Virus Recovery in Chicken Cells Tested with Rous Sarcoma Cell DNA

DNA from non-virus-producing RSV transformed mammalian cells converts chicken fibroblasts into Rous sarcoma cells producing infectious RSV particles. The recovered virus is the same biologically and antigenically as the virus which originally transformed the mammalian cells.     

Low resistance junctions between normal and between virus transformed fibroblasts in tissue culture

The occurrence of low resistance junctions between normal chick embryo fibroblasts and between fibroblasts transformed with Rous sarcoma virus in tissue culture was studied with intracellular microelectrodes. The results showed that these junctions were present between normal chick fibroblasts in proliferating cultures as well as between cells in ‘density dependent inhibited cultures’. Mechanical injury to a fibroblast within a small group of coupled cells caused the injured fibroblast to uncouple immediately from its neighboring cells without interrupting coupling between the healthy uninjured cells. In the case of fibroblasts transformed with a Rous sarcoma virus, the results further showed that low resistance junctions were present when the transformation appeared in the infected cells and remained present thereafter.     

Reduced binding of epidermal growth factor by avian sarcoma virus-transformed rat cells

Rat cells transformed by Rous sarcoma virus and Fujinami sarcoma virus bound 5-10% of the amount of epidermal growth factor (EGF) bound by normal cells. Scatchard plot analysis indicated that the reduction in binding by transformed cells was due to a decreased number of receptors rather than to altered binding affinity. In experiments with temperature sensitive mutants of Rous sarcoma virus and Fujinami sarcoma virus significant loss of EGF binding occurred within one hour of shift from non-permissive to permissive temperature. Conditioned media from various normal and transformed cell lines were examined for the ability to inhibit EGF binding to normal cells or to cause "down regulation" of EGF receptors. No activity of either type was found. EGF-dependent phosphorylation in isolated membrane preparations was also examined. Membranes from normal cells displayed EGF-dependent phosphorylation of a Mr 180,000 protein presumed to be the EGF receptor. This activity was absent in membranes from transformed cells. The data suggest a close correlation between activation of avian sarcoma virus transforming gene products and modulation of the EGF growth regulatory system.     

Immunological and structural properties of Rous sarcoma virus-transformed and tumor cells]

We compared the capacity of both normal and Rous sarcoma virus (RSV)-transformed chicken embryo fibroblast (CEF) cells as well as Rous sarcoma (RS) tumor cells to serve as targets in anti-tumor immunity assays. These studies showed that sera from tumor-bearing donors were able to stain transformed CEF more efficiently than RS cells as detected by immunofluorescence. In contrast, antiserum against the major viral glycoprotein, gp 85, stained a higher percentage of RS than transformed cells. Normal CEF cells, which served as controls, were essentially non-reactive with the immune system as judged by this type of assay. We observed that RS cells are considerably larger and contain far higher levels of protein than either normal or transformed CEF. Scanning electron microscopy revealed both the RS cells and transformed CEF to be rich in surface ruffles, blebs and microvilli as distinguished from the flattened, fusiform appearance of normal CEF cells.     

Production of Virus by Mammalian Cells Transformed by Rous Sarcoma and Murine Sarcoma Viruses

Cultured cells of mammalian tumors induced by ribonucleic acid (RNA)-containing oncogenic viruses were examined for production of virus. The cell lines were established from tumors induced in rats and hamsters with either Rous sarcoma virus (Schmidt-Ruppin or Bryan strains) or murine sarcoma virus (Moloney strain). When culture fluids from each of the cell lines were examined for transforming activity or production of progeny virus, none of the cell lines was found to be infectious. However, electron microscopic examination of the various cell lines revealed the presence of particles in the rat cells transformed by either Rous sarcoma virus or murine sarcoma virus. These particles, morphologically similar to those associated with murine leukemias, were found both in the extracellular fluid concentrates and in whole-cell preparations. In the latter, they were seen budding from the cell membranes or lying in the intercellular spaces. No viruslike particles were seen in preparations from hamster tumors. Exposure of the rat cells to (3)H-uridine resulted in the appearance of labeled particles with densities in sucrose gradients typical of virus (1.16 g/ml.). RNA of high molecular weight was extracted from these particles, and double-labeling experiments showed that this RNA sedimented at the same rate as RNA extracted from Rous sarcoma virus. None of the hamster cell lines gave radioactive peaks in the virus density range, and no extractable high molecular weight RNA was found. These studies suggest that the murine sarcoma virus produces an infection analogous to certain "defective" strains of Rous sarcoma virus, in that particles produced by infected cells have a low efficiency of infection. The control of the host cell over the production and properties of the RNA-containing tumorigenic viruses is discussed.     

Appearance of virus-specific DNA in mammalian cells following transformation by Rous sarcoma virus

To detect Rous sarcoma virus-specific DNA in mammalian cells, we have measured the capacity of unlabeled cell DNA to accelerate the reassociation of labeled double-stranded DNA synthesized by the Rous sarcoma virus RNA directed DNA polymerase. Two populations of double-stranded polymerase products are identified by their reassociation kinetics and represent approximately 5% and 30% of the viral 70 S RNA genome. Using two strains of Rous sarcoma virus and four lines of transformed mammalian cells, we found two copies of DNA homologous to both DNA populations in Rous sarcoma virustransformed rat and mouse cells, but not in normal cells. The Rous sarcoma viruslike DNA can be demonstrated in the non-repeated fraction of transformed cell DNA and in nuclear DNA. The results are supported by evidence that the techniques employed detect the formation of extensive well-matched duplexes of cell DNA and viral polymerase products.     

The effect of microtubule disruption on the distribution of the cytoskeletal proteins, vinculin, alpha-actinin, and actin in normal and RSV-transformed fibroblasts

By double indirect immunofluorescence and interference electron microscopy, we have observed the effect of microtubule disruption by antimitotic drugs and coldness treatment on the distribution of adhesion sites and of the three cytoskeletal proteins, vinculin, alpha-actinin, and actin in normal rat cells and in rat cells transformed by Rous sarcoma virus. This study shows that the state of organization of the microtubule--intermediate filament complex modulates the location and the arrangement of intracellular structures containing vinculin, alpha-actinin, and actin in normal as well as in transformed cells. The most important alterations are observed in transformed cells on the distribution of the rosette clusters that have been shown to characterize the transformation by Rous sarcoma virus [8]. These results suggest the microtubule-intermediate filament complex is directly or indirectly connected with the microfilament network.     

Four different classes of retroviruses induce phosphorylation of tyrosines present in similar cellular proteins.

Chicken embryo cells transformed by the related avian sarcoma viruses PRC II and Fujinami sarcoma virus, or by the unrelated virus Y73, contain three phosphoproteins not observed in untransformed cells and increased levels of up to four other phosphoproteins. These same phosphoproteins are present in increased levels in cells transformed by Rous sarcoma virus, a virus which is apparently unrelated to the three aforementioned viruses. In all cases, the phosphoproteins contain phosphotyrosine and thus may be substrates for the tyrosine-specific protein kinases encoded by these viruses. In one case, the site(s) of tyrosine phosphorylation within the protein is the same for all four viruses. A homologous protein is also phosphorylated, at the same major site, in mouse 3T3 cells transformed by Rous sarcoma virus or by the further unrelated virus Abelson murine leukemia virus. A second phosphotyrosine-containing protein has been detected in both Rous sarcoma virus and Abelson murine leukemia virus-transformed 3T3 cells, but was absent from normal 3T3 cells and 3T3 cells transformed by various other viruses. We conclude that representatives of four apparently unrelated classes of transforming retroviruses all induce the phosphorylation of tyrosines present in the same set of cellular proteins.     

Potassium fluxes and ouabain binding in growing,density-inhibited and rous sarcoma virus-transformed chicken embryo cells

Potassium fluxes, ouabain binding, and Na⁺ and K⁺ intracellular concentrations were determined for cultures of growing normal, density-inhibited and Rous sarcoma virus-transformed chicken embryo fibroblasts. No significant differences in K⁺ influx or ouabain binding were detected between growing normal cells and Rous sarcoma virus-transformed cells; however, ouabain binding and ouabain-sensitive K⁺ influx were 1.5- to 1.8-fold lower in density-inhibited cells. Thus, potassium influx in this system can be classified as a growth-related, but not transformation-specific change. As determined by both flame photometry and radioisotopic (⁴²K) equilibration, growing normal and density-inhibited cells had similar potassium contents, whereas transformed cells exhibited 1.4-fold higher potassium levels. Sodium ion levels, as measured by flame photometry, were also 2- to 4.5-fold higher in transformed than normal or density-inhibited cells. Complementary studies of potassium efflux showed a 1.3- to 1.5-fold higher rate (based on the percentage of pool exiting the cell) in growing normal versus density-inhibited or transformed fibroblasts. Because of the larger potassium pool in transformed cells, efflux based on absolute number of potassium ions is similar in normal and transformed chicken embryo fibroblasts.     

Modification of gap junctions in cells transformed by a temperature-sensitive mutant of Rous sarcoma virus

Summary Prompted by our observation that a reduction in junctional permeance is one of the earlier events in the process of neoplastic transformation of a cell line by Rous sarcoma virus, we analyzed the gap junctions, from these cells to determine if the basis of the reduction is a loss of junctional channels. The cells (normal rat kidney, or NRK) are infected with a temperature-sensitive mutant of Rous sarcoma virus, allowing one easily to manipulate the cells into and out of the transformed state, and hence also to manipulate the junctional permeance. Using freeze-fracture electron microscopy, we found that the number and size of the junctions did not change in parallel with the permeance changes we had previously characterized. There is, however, a significant rearrangement of the junctional particles to a more random configuration when the cells are transformed and a reversal to the more ordered pattern when the cells are shifted back to the normal phenotype. These changes do parallel the changes in junctional permeance. We conclude that the permeance of existing junctional channels is modified and that the change in permeance may involve a change in the interaction of the junctional channels with each other and/or the surrounding lipid domain.     

Comparison of the expression of the src gene of Rous sarcoma virus in vitro and in vivo.

We have compared the polypeptide products of the src gene of several strains of Rous sarcoma virus produced by in vitro translation of heat-denatured 70S virion RNA in the nuclease-treated reticulocyte lysate with those present in chick cells transformed by these viruses. We have done this by immunoprecipitation, using sera from rabbits injected at birth with Schmidt-Ruppin Rous sarcoma virus. In vitro translation results in the synthesis of at least nine polypeptides which appear to be encoded by the src gene. These range in size from 17,000 to 60,000 daltons. The sera from tumor-bearing rabbits precipitated these polypeptides arising from the in vitro translation of RNA from Schmidt-Ruppin Rous sarcoma virus of both subgroup A and subgroup D and from one stock of Prague Rous sarcoma virus of subgroup C. In each case, all of this family of related polypeptides could be precipitated except the smallest, the 17,000-dalton polypeptide. No precipitation of analogous polypeptides resulting from the translation of RNA from other strains of Rous sarcoma virus was observed. Cells transformed by these three strains of Rous sarcoma virus contain easily detectable amounts of a polypeptide, p60src, essentially identical to the 60,000-dalton in vitro product. With one exception, they do not contain significant amounts of polypeptides analogous to the smaller in vitro products which can be precipitated by these sera. Cells transformed by one stock of Schmidt-Ruppin Rous sarcoma virus of subgroup A did contain a 39,000-dalton polypeptide, which was related, by peptide mapping, to the 60,000-dalton polypeptide and was similar in size to a precipitable in vitro product. The 60,000-dalton polypeptide present in transformed cells appeared to be phosphorylated 10 to 25 min after its synthesis, metabolically very stable, and not derived from a precursor polypeptide. All immunoprecipitates from transformed cells which contained p60src also contained an 80,000-dalton phosphoprotein. This polypeptide is unrelated to p60src, as determined by peptide mapping, and may well be a host cell polypeptide which is specifically associated with p60src.     

The same normal cell protein is phosphorylated after transformation by avian sarcoma viruses with unrelated transforming genes.

The phosphorylation of a normal cellular protein of molecular weight 34,000 (34K) is enhanced in Rous sarcoma virus-transformed chicken embryo fibroblasts apparently as a direct consequence of the phosphotransferase activity of the Rous sarcoma virus-transforming protein pp60src. We have prepared anti-34K serum by using 34K purified from normal fibroblasts to confirm that the transformation-specific phosphorylation described previously occurs on a normal cellular protein and to further characterize the nature of the protein. In this communication, we also show that the phosphorylation of 34K is also increased in cells transformed by either Fujinami or PRCII sarcoma virus, two recently characterized avian sarcoma viruses whose transforming proteins, although distinct from pp60src, are also associated with phosphotransferase activity. Moreover, comparative fingerprinting of tryptic phosphopeptides shows that the major site of phosphorylation of 34K is the same in all three cases.     

The response of chicken embryo dermal fibroblasts to cytochalasin B is altered by Rous sarcoma virus-induced cell transformation.

The drug cytochalasin B (CB), which disrupts the cellular microfilament network, allows the identification of as yet unclassified structural differences between normal and Rous sarcoma virus-transformed chicken embryo fibroblasts. When exposed to CB, normal chick fibroblasts attain an arborized or dendritic morphology. This results as the cytoplasm collapses upon the remaining structural and adhesive components of the cell. Rous sarcoma virus-transformed cells did not form or maintain these dendritic-like processes in the presence of CB and, as a result, rounded up but still remained attached to the substrate. With a temperature-sensitive mutant of Rous sarcoma virus, LA24A, it was possible to show that these effects are completely reversible and dependent on the expression of pp60src. The cytoskeleton in these CB-treated cells was examined by both immunofluorescence and electron microscopy. After exposure to CB, the microfilaments were found to be disrupted similarly throughout both the transformed and the nontransformed cells. In the nontransformed cells arborized by exposure to CB, the extended processes were found to contain intermediate filaments in an unusually high concentration and degree of organization. The distribution of these filaments in the central body of the arborized cells was random. This lower concentration and random distribution was similar to that seen throughout the transformed cells rounded up by exposure to CB. The failure of these transformed cells to arborize in CB indicates that the structural component(s) which is necessary for the formation or maintenance or both of the arborized state is altered by the expression of pp60src.     

Effect of microinjected amine and diamine oxidases on the ultrastructure of eukaryotic cultured cells

Diamine oxide and serum amine oxidase, which catalyse the oxidation of diamines and polyamines, respectively, were trapped within reconstituted Sendai virus envelopes. These loaded envelopes were incubated with cultured normal chick fibroblasts or with fibroblasts transformed by Rous sarcoma viruses. The binding of the reconstituted envelopes to the cultured cells was confirmed by scanning electron microscopy. It has been shown that the reconstituted envelopes (1-3 microns diameter) were attached to the eukaryotic cells. No significant changes in the morphology of the normal chick embryo fibroblasts were noted upon treatment with enzyme-loaded envelopes. On the other hand, chick embryo fibroblasts transformed by Rous sarcoma virus were affected by the microinjected amine oxidases. Scanning electron microscopy demonstrated the formation of holes in the microinjected cells. Similar morphological changes were also observed when diamine oxidase was microinjected into cultured glioma cells. These holes may be the result of the ejection of the nucleus. These findings are in line with the observed effect of the injected amine oxidases on macromolecular synthesis in normal and transformed chick embryo fibroblasts.     

Changes in the synthesis and phosphorylation of cellular proteins in chick fibroblasts transformed by two avian sarcoma viruses

35S- and 32P-labeled proteins from control chick embryo fibroblasts and from fibroblasts transformed by UR2 sarcoma virus, or by a temperature-sensitive mutant (tsLA29) of Rous sarcoma virus, were separated by two-dimensional electrophoresis on giant gels to detect transformation-specific changes in protein synthesis and total phosphorylation. A nontransforming avian retrovirus, UR2-associated virus (UR2AV), was also studied. Virus-coded proteins appear in whole cell lysates of all infected cells. The structural proteins can be identified by comparison with proteins immunoprecipitated with antivirus serum. The transforming proteins pp60src and p68ros, present in cells transformed with Rous sarcoma virus and UR2, respectively, are phosphorylated in vivo. Eighteen increases and eight decreases in cellular phosphoproteins are associated with transformation, and revert toward normal levels when cells infected with tsLA29 are incubated at 42 degrees C. These changes are more extensive than previously reported, but none represent new phosphorylations, since all phosphoproteins seen in transformed cells also appear to be phosphorylated to a certain extent in control cells. Fifteen cellular proteins show increased relative rates of synthesis apparently related either to transformation or to growth at 42 degrees C. Four other proteins are increased exclusively in cells incubated at 42 degrees C, but not at 37 degrees C, whether transformed or not. Eleven additional increases in the synthesis of cellular proteins, many quite large, and one seemingly a de novo induction, appear to be specific for transformation. These changes occur in cells transformed by either UR2 or Rous sarcoma virus at 37 degrees C, do not occur with UR2AV infection, and tend to revert in cells infected with tsLA29 incubated at 42 degrees C. These 11 changes may represent increases in cellular gene expression that are related specifically to the maintenance of the transformed state.     

Membrane lipid dynamics and density dependent growth control in normal and transformed avian cells

Membrane fluidity of normal chick embryo fibroblasts and normal Japanese quail fibroblasts and their Rous sarcoma virus and methylcholanthrene transformed counterparts was investigated using the technique of fluorescence depolarisation of 1,6-diphenylhexatriene incorporated in the whole cells and in their isolated plasma membrane vesicles. Normal cells and isolated plasma membranes of normal cells showed significant changes in fluidity as a function of population density while neither Rous sarcoma virus transformed nor methylcholanthrene tumor cells or their isolated plasma membrane showed this effect. Stimulation of growth by addition of calf serum to cultures of quiescent, density-inhibited normal cells was accompanied by rapid changes in the direction of increased membrane lipid fluidity. Neither sparse normal cells, nor sparse or dense transformed cells showed any significant fluidity change in their membrane lipids upon addition of serum. Enzyme and electron microscopic analysis of the ratios of different membrane types in each cell type showed that this ratio was invariant with respect to cell population density but different between transformed and normal cells. Hence, the fluidity changes observed, measured as the mean rotational correlation time of the fluorescene probe in the membrane lipids, truly reflect organisational differences, occurring as a function of population density in cultures of cells which retain density-dependent growth control.     

The uncoupled regulation of fibronectin and collagen synthesis in Rous sarcoma virus transformed avian tendon cells.

We have investigated the regulation of fibronectin and procollagen synthesis in normal and Rous sarcoma virus transformed primary avian tendon cells. These two proteins interact at the cell periphery and both are reportedly lost upon transformation. We thus examined whether their synthesis was coordinately regulated in Rous sarcoma virus-infected cells. It was found that while the synthesis of both pro alpha 1 and pro alpha 2 peptides was reduced upon transformation, the synthesis of fibronectin was not altered. Nevertheless, long term radiolabeling demonstrated that fibronectin levels were reduced in transformed cells. It is concluded that the reduction in levels of these components at the surface is brought about by different mechanisms; collagen levels being regulated by procollagen synthesis and fibronectin levels by degradation and/or release into the culture medium. The possibility is discussed that fibronectin is lost from the cell periphery of primary avian tendon cells as a consequence of decreased levels of anchoring collagen molecules.     

Shedding of hyaluronate from the cell surface of Rous sarcoma virus-transformed chondrocytes.

Transformation of cultured chick embryo chondrocytes with Rous sarcoma virus gives rise to increased incorporation of isotopic precursors into hyaluronate and decreased incorporation into chondroitin 6-sulfate. Chemical measurements of these glycosaminoglycans showed corresponding changes. Comparison of the kinetics of production of glycosaminoglycan by normal and Rous sarcoma virus-transformed chondrocytes demonstrated (i) that the rate of accumulation in the medium was similar in both cultures, and (ii) that approximately 50% of total glycosaminoglycan produced by the normal chondrocytes, but only 10% of that from the transformed cells, accumulated in the cell layer. Prelabel-chase experiments indicated that cell surface-associated hyaluronate, as measured by release from the cell layer by trypsin treatment, was shed rapidly into the medium and accounted for all of the hyaluronate which accumulated in the medium. Thus we conclude (i) that accumulation of cell surface-associated glycosaminoglycan is dramatically reduced in Rous sarcoma virus-transformed chondrocytes, and (ii) that hyaluronate produced by the transformed chondrocytes is first deposited in the cell-associated extracellular compartment and then rapidly shed into the medium, rather than being secreted directly into the medium.     

31P NMR spectra of rodent and avian fibroblasts transformed by Rous or Kirsten sarcoma viruses

31P NMR spectra of normal rodent and avian fibroblasts were compared to those of the same cells transformed either by the Rous sarcoma virus (RSV) or by the Kirsten sarcoma virus (Ki-MSV). Under physiological conditions, the spectra of living or perchloric acid extracted chicken embryo fibroblasts, rat cell line FR3T3 and mouse cell line C127 did not differ from those of their counterparts transformed by RSV or Ki-MSV. However, in the case of FR3T3 cells, on shifting from 37 degrees C to 20 degrees C, and particularly if PBS replaced serum growth medium, a different, though transitory, response of the transformed cells was detected. They then showed, within few minutes, a more rapid ATP depletion with accumulation of fructose 1,6-diphosphate (FDP), as compared to normal control cells.